Cytotoxic Polyprenylated Xanthones from the Resin of Garcinia

Dec 12, 2008 - A geranyl and a prenyl group were assigned to C-2 and C-4, ... (3) Partial structure b was determined by the 1H−1H COSY and ... 16, 5...
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J. Nat. Prod. 2009, 72, 117–124

117

Cytotoxic Polyprenylated Xanthones from the Resin of Garcinia hanburyi Si-Jia Tao,† Shu-Hong Guan,† Wei Wang,‡ Zhi-Qiang Lu,† Guang-Tong Chen,† Na Sha,† Qing-Xi Yue,† Xuan Liu,† and De-An Guo*,† Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Guo Shoujing Road 199, Zhangjiang, Shanghai 201203, People’s Republic of China, and National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy, School of Pharmacy, UniVersity of Mississippi, Mississippi 38677 ReceiVed July 23, 2008

Twelve new xanthones (1-12), a pair of new natural products (13 and 14), and 18 known related compounds were isolated from the resin of Garcinia hanburyi. The structures of 1-14 were elucidated by detailed spectroscopic analyses. A cytotoxic assay of the isolated compounds revealed that, with the exception of 2, these compounds were active against the HeLa tumor cell line. Gamboge is the dried resin most often exuded from the stems of Garcinia hanburyi Hook. f. (Guttiferae), which is found throughout Thailand, Cambodia, India, and southern China. It is used as a dye or folk medicine for an internal purgative and externally infected wounds.1 Previous phytochemical analyses of G. hanburyi led to the isolation of more than 30 caged polyprenylated xanthones.2-8 In our continuing search for biologically active and structurally unique compounds from medicinal plants, we have chemically investigated the resin of G. hanburyi. Twelve new polyprenylated xanthones (1-12), a pair of new natural products (13 and 14), and 18 known xanthones were isolated from the resin, including the known gambogic acid as a major active constituent.9-12 Herein, we report the isolation and structural elucidation of compounds 1-14, as well as the assessment of their cytotoxicity against HeLa tumor cells.

Results and Discussion Compound 1 was obtained as a yellow gum. Its molecular formula was determined to be C37H46O7, established by HRESIMS ([M + Na]+ at m/z 625.3138, calcd 625.3141). Its 1H and 13C NMR spectra, in conjunction with the HSQC spectrum, revealed the presence of eight methyl, six methylene, eight methine, and 15 quaternary carbons. The NMR data of 1 (Tables 1 and 2) did not indicate much similarity to those of the caged pentaprenylxanthonoids such as gambogic acid (C38H44O8), which was previously isolated from G. hanburyi.2 The presence of a ketal carbon (δC 107.6) was similar to that of gaudispirolactone, a highly rearranged tetraprenylxanthonoid isolated from the bark of Garcinia gaudichaudii.13 The aforementioned features suggested that 1 was a degraded and rearranged pentaprenylxanthonoid. Interpretation of the 1H-1H COSY and HMBC spectra afforded two subunits (a, b) (Figure 1). In the partial structure a, the HMBC correlations between a hydrogen-bonded hydroxy proton (δH 12.21) and C-1, C-2, and C-9a and between a hydroxy proton (δH 6.50) and C-2, C-3, and C-4 indicated the location of the two hydroxy groups at C-1 and C-3. A geranyl and a prenyl group were assigned to C-2 and C-4, respectively, which was confirmed by the HMBC correlations of H-11/C-1, C-2, and C-3 and H-21/C-3, C-4, and C-4a. Thus, the structure of subunit a was established, which was identical to the partial structure of gambogenic acid.3 Partial structure b was determined by the 1H-1H COSY and HMBC data. In the HMBC spectrum, the correlations of H-27/C-26, C-28, and * To whom correspondence should be addressed. Tel: 86-21-50271516. Fax: 86-21-50272789. E-mail: [email protected]. † Shanghai Institute of Materia Medica. ‡ University of Mississippi.

10.1021/np800460b CCC: $40.75

C-29, H-28/C-26 and C-27, and H-31/C-28, C-29, and C-30 suggested the presence of an R,β-unsaturated δ-lactone moiety. The 1H-1H COSY experiment indicated a contiguous spin system comprising H-8a/H-8/H-7/H-6/H-5. In conjunction with the HMBC correlations of H-5/C-8a and C-10a, it showed the presence of a six-membered ring. In addition, the HMBC correlations of H-5/C10a, C-26, C-32, C-33, and C-34, H-33/C-32, and H-34/C-32 suggested the presence of a five-membered ring, hence completing the partial structure b, which was similar to that of gaudispirolactone.13 The only difference between these two partial structures was that the R,β-unsaturated γ-lactone moiety in gaudispirolactone was replaced by a cis-double bond in 1. The pentaprenylxanthonoid structure was finally established by combining the two partial structures. The HMBC correlation from H-8 to C-9 permitted the linkage of subunits a and b. Scheme 1 outlined a postulated biosynthesis pathway of 1. The relative configuration of 1 was assessed by analyzing the ROESY spectrum and a computergenerated 3D structure, which was obtained by Chem 3D Ultra V 9.0, with MM2 forcefield calculations for energy minimization (Figure 2). Key ROESY correlations were observed between H-8a and H-27, H-33, and between H-5 and H-6a, H-34. Thus, the relative configuration of 1 was elucidated as shown in Figure 2, and the structure was established as gambospiroene. Compound 2 was isolated as a yellow gum. Its molecular formula, C34H40O8, was established on the basis of HREIMS ([M]+ at m/z 576.2708, cald 576.2723), with 15 degrees of unsaturation. The UV absorption bands at λmax 366, 314, 275, and 266 nm were similar to those of moreollic acid (2a).3 The 1H NMR spectrum of 2 (Table 1) revealed the presence of a hydrogen-bonded hydroxy group [δH 11.96 (1H, s, 1-OH)], two prenyl olefinic protons [δH 5.02 (1H, t, J ) 7.2 Hz), δH 6.45 (1H, t, J ) 7.2 Hz)], a cis-double bond [δH 5.50 (1H, d, J ) 10.0 Hz), δH 6.61(1H, d, J ) 10.0 Hz)], a methoxy group [δH 3.67 (3H, s)], and seven methyl groups [δH 1.13, 1.34, 1.39, 1.45, 1.64, 1.75, 1.93 (each 3H, s)]. The aforementioned features suggested that 2 had a caged polyprenylated xanthone moiety without a C8-C8a double bond.4 In the 13C NMR spectrum, the upfield shift of C-24 (-3.1 ppm), compared to 2a, and the HMBC correlations of O-methyl protons with C-24 suggested the location of a methoxy group at C-24. Additionally, the 13C NMR data of 2 showed one more CH2 and one less CH than in 2a. This, in conjunction with the HMBC correlation of H28/C-26, supported the presence of a methylene at C-8. From the ROESY cross-peak between H-22 (δH 6.45) and H-25 (δH 1.93), the olefinic bond of this prenyl group had a Z-configuration. Therefore, the structure of 2 was established as methyl 8,8adihydromorellate.

 2009 American Chemical Society and American Society of Pharmacognosy Published on Web 12/12/2008

a

1

1.13 s 1.45 s

1.70 s

2.54 dd (2.0, 4.0) 6.18 m

1.66 s 1.58 s 1.78 s 3.20, 3.33 m 5.16 t (7.2) 1.77 s 1.69 s

6.50 s 3.25 d (8.8) a 2.45 m b 2.13 m 5.99 m 5.56 m 3.17b 3.30b 5.18 t (7.2) 2.05 m 2.08 m 5.04 t (6.8)

12.21 s

2

3.67 s

1.13 s 1.34 s

1.93 s a 1.96 m b 1.54 m 2.58 d (8.4)

1.75 s 1.64 s 3.18, 3.25 m 6.45 t (7.2)

2.42 m 1.71, 2.84 m 3.22b 6.61 d (10) 5.50 d (10) 1.39 s 1.45 s 3.16, 3.30 m 5.02 t (7.2)

11.96 s

1.28 s 1.71 s

0.99 s 1.37 s 2.34 dd (4.4, 13.2) 1.35 dd (9.6, 13.2) 2.49 d (9.2)

4.40 dt (1.2, 7.2)

1.68 s 1.61 s 1.72 s 3.33 m 5.20 dt (1.2, 7.2) 1.72 s 1.66 s 2.58 d (7.6)

1.28 s 1.69 s

1.71 s 2.30 dd (4.8, 13.6) 1.37 dd (9.2, 13.2) 2.49 d (9.6)

6.12 t (6.8)

1.43 s 1.08 s 1.65 s 3.10, 3.30 m 4.97 m 1.71 s 1.60 s 2.85, 3.00 m

3.18 m 6.34 br s 2.14 m 1.38, 1.88 m 1.62 m

4

4.59 d (6.8) 5.44 dt (1.2, 7.2) 2.09 m 2.11 m 5.09 dt (1.2, 7.2)

13.31 s

3.47 dd (4.4, 6.4) 7.55 d (7.2)

3

3.49 dd (4.4, 6.8) 7.45 d (7.2)

12.76 s 6.10 s

5

1.31 s 1.69 s 3.63 s

1.72 s 2.31 d (12.8) 1.66b 2.60 d (9.2)

6.04 t (7.2)

4.20, 4.55 s 1.85 s 1.33 s 3.11, 3.26 m 5.05 t (6.8) 1.72 s 1.64 s 3.00 d (7.6)

3.42 m 1.73, 1.78b 1.54, 1.90 m 1.26, 1.38 m 2.12 m

7.56 s

12.58 s

6

1.30 s 1.67 s 3.62 s

1.73 s 2.31 d (12.8) 1.66b 2.58 d (9.2)

6.13 t (7.2)

1.64 s 1.53 s 1.36 s 3.11, 3.28 m 5.00b 1.71 s 1.61 s 3.00 d (7.2)

6.56 d (10.4) 5.37 d (10.4) 1.56, 1.72 m 2.00 m 5.03b

7.61 s

12.67 br s

Recorded at 400 MHz in CDCl3. δH in ppm, J in Hz. b J were omitted, as the signals were overlapped.

27 28 29 30 31a 31b 32 33 34 35 7-OCH3 24-OCH3

1-OH 2 3-OH 5 6 7 8 8a 11 12 14 15 16 17 18 19 20 21 22 24 25 26

position

Table 1. 1H NMR Data for Compounds 1-12a 7

1.30 s 1.68 s 3.62 s

1.74 s 2.30 d (12.8) 1.66b 2.57 d (9.2)

6.17 t (7.2)

1.66 s 1.57 s 1.32 s 3.12, 3.30 m 5.00 t (6.4) 1.72 s 1.62 s 2.98 m

6.56 d (10.4) 5.40 d (10.4) 1.64, 1.77 m 2.07 m 5.09 t (7.2)

7.63 s

12.69 s

8

1.12 s 1.33 s

1.93 s a 1.90b b 1.38b 2.43 d (8.4)

1.73 s 1.62 s 3.20 m 6.58 t (6.8)

2.66 m 4.80 d (3.6) 3.22b 6.58 d (9.6) 5.50 d (9.6) 1.38 s 1.44 s 3.15, 3.25 m 4.97 t (6.8)

11.87 br s

9

1.09 s 1.30 s

1.89 s 1.86b 1.34b 2.39 d (8.0)

6.61 t (6.8)

1.62 s 1.55 s 1.75 s 3.26 m 5.08 t (6.8) 1.76 s 1.70 s 3.10, 3.30 m

2.67 m 4.77 d (4.0) 3.16b 3.20b 5.18 t (6.8) 2.00 m 2.04 m 5.02 t (7.0)

6.51 brs

11.88 br s

10

1.28 s 1.68 s

1.69 s 2.34 dd (4.8, 13.6) 1.38 dd (9.2, 13.6) 2.50 d (10)

5.72 t (7.2)

1.28 s 1.22 s 1.44 s 3.26 m 5.09 t (6.4) 1.75 s 1.65 s 2.92, 3.20 m

6.63 d (10) 5.32 d (10) 2.26, 2.48 m 5.63b 5.63b

3.50 m 7.50 d (7.2)

12.78 s

11

1.29 s 1.66 s

1.72 s 2.31 dd (4.2, 13.2) 1.37 dd (10, 13.2) 2.45 d (9.2)

5.67 m

1.64 s 1.55 s 1.69 s 2.93, 3.10 m 4.55 t (8.8) 1.14 s 1.23 s 2.82, 3.42 m

3.05, 3.22 m 5.15 t (7.2) 1.92 m 2.00 m 5.05 t (6.8)

3.50 m 7.49 d (6.8)

12.83 s

12

3.63 s

1.31 s 1.70 s

1.76 s 1.67 s 2.64 m 4.78 m 1.06 s 3.66b a 2.37 d (13.2) b 1.62b 2.58 d (9.6)

6.64 d (10) 5.53 d (10) 1.44 s 1.44 s 3.30 m 5.20 m

7.47 s

12.63 br s

118 Journal of Natural Products, 2009, Vol. 72, No. 1 Tao et al.

a

2

51.3

156.0 (qC) 102.3 (qC) 160.5 (qC) 108.8 (qC) 155.8 (qC) 86.5 (qC) 210.7 (qC) 38.6 (CH) 21.8 (CH2) 39.1 (CH) 195.6 (qC) 102.8 (qC) 88.7 (qC) 115.4 (CH) 126.2 (CH) 78.3 (qC) 28.2 (CH3) 28.5 (CH3) 21.5 (CH2) 122.7 (CH) 131.0 (qC) 18.0 (CH3) 25.6 (CH3) 28.1 (CH2) 137.4 (CH) 127.5 (qC) 168.2 (qC) 20.7 (CH3) 23.5 (CH2) 44.4 (CH) 82.2 (qC) 27.3 (CH3) 29.7 (CH3)

Recorded at 100 MHz in CDCl3.

1

159.3 (qC) 107.3 (qC) 163.7 (qC) 106.6 (qC) 155.3 (qC) 44.3 (CH) 21.2 (CH2) 128.4 (CH) 121.4 (CH) 44.6 (CH) 195.5 (qC) 101.3 (qC) 83.7 (qC) 21.1 (CH2) 121.4 (CH) 138.7 (qC) 39.7 (CH2) 26.4 (CH2) 123.8 (CH) 131.9 (qC) 25.7 (CH3) 17.7 (CH3) 16.2 (CH3) 21.8 (CH2) 121.7 (CH) 133.9 (qC) 17.9 (CH3) 25.8 (CH3) 107.6 (qC) 29.6 (CH2) 135.2 (CH) 127.6 (qC) 164.2 (qC) 16.5 (CH3) 82.6 (qC) 24.1 (CH3) 31.6 (CH3)

3 163.6 (qC) 93.5 (CH) 165.7 (qC) 108.8 (qC) 157.3 (qC) 84.7 (qC) 203.6 (qC) 46.9 (CH) 133.8 (CH) 133.8 (qC) 179.6 (qC) 100.7 (qC) 90.3 (qC) 65.7 (CH2) 118.6 (CH) 141.8 (qC) 39.4 (CH2) 26.2 (CH2) 123.6 (CH) 131.9 (qC) 25.7 (CH3) 17.7 (CH3) 16.7 (CH3) 21.8 (CH2) 122.2 (CH) 131.8 (qC) 18.1 (CH3) 25.8 (CH3) 28.8 (CH2) 117.9 (CH) 135.0 (qC) 16.7 (CH3) 25.5 (CH3) 25.5 (CH2) 49.2 (CH) 83.2 (qC) 29.1 (CH3) 30.1 (CH3)

NMR Data for Compounds 1-12a

position

13C

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 7-OCH3 24-OCH3

Table 2. 4 161.5 (qC) 105.3 (qC) 162.3 (qC) 108.1 (qC) 155.0 (qC) 84.0 (qC) 203.5 (qC) 46.7 (CH) 135.0 (CH) 133.5 (qC) 178.9 (qC) 100.2 (qC) 90.4 (qC) 32.8 (CH) 123.4 (CH) 133.7 (qC) 31.1 (CH2) 24.8 (CH2) 45.0 (CH) 80.0 (qC) 27.4 (CH3) 19.9 (CH3) 23.3 (CH3) 21.8 (CH2) 122.5 (CH) 131.1 (qC) 18.1 (CH3) 25.8 (CH3) 29.3 (CH2) 137.2 (CH) 127.6 (qC) 170.8 (qC) 20.8 (CH3) 25.2 (CH2) 48.9 (CH) 83.8 (qC) 28.8 (CH3) 29.8 (CH3)

5 160.8 (qC) 104.2 (qC) 164.6 (qC) 106.3 (qC) 155.1 (qC) 83.8 (qC) 202.2 (qC) 84.6 (qC) 134.5 (CH) 132.3 (qC) 177.8 (qC) 99.6 (qC) 89.1 (qC) 28.8 (CH) 36.4 (CH2) 77.2 (qC) 39.2 (CH2) 22.7 (CH2) 48.0 (CH) 147.8 (qC) 108.6 (CH2) 23.0 (CH3) 28.4 (CH3) 21.8 (CH2) 122.2 (CH) 131.2 (qC) 18.1 (CH3) 25.8 (CH3) 29.1 (CH2) 137.9 (CH) 127.9 (qC) 171.2 (qC) 20.7 (CH3) 30.2 (CH2) 49.4 (CH) 84.3 (qC) 28.8 (CH3) 30.0 (CH3) 54.1

6 157.4 (qC) 102.7 (qC) 161.6 (qC) 107.6 (qC) 157.1 (qC) 83.4 (qC) 201.9 (qC) 84.7 (qC) 135.4 (CH) 131.7 (qC) 178.1 (qC) 100.4 (qC) 89.6 (qC) 115.7 (CH) 124.5 (CH) 81.4 (qC) 41.9 (CH2) 22.7 (CH2) 123.7 (CH) 131.8 (qC) 25.6 (CH3) 17.6 (CH3) 27.7 (CH3) 21.5 (CH2) 122.0 (CH) 131.6 (qC) 18.0 (CH3) 25.6 (CH3) 29.1 (CH2) 138.4 (CH) 127.6 (qC) 171.7 (qC) 20.7 (CH3) 30.2 (CH2) 49.4 (CH) 84.2 (qC) 28.8 (CH3) 29.9 (CH3) 54.1

7 157.5 (qC) 102.9 (qC) 161.5 (qC) 107.8 (qC) 157.1 (qC) 83.3 (qC) 201.9 (qC) 84.7 (qC) 135.6 (CH) 131.5 (qC) 178.2 (qC) 100.6 (qC) 89.7 (qC) 115.8 (CH) 124.8 (CH) 81.2 (qC) 41.6 (CH2) 22.7 (CH2) 123.8 (CH) 132.0 (qC) 25.6 (CH3) 17.6 (CH3) 26.9 (CH3) 21.6 (CH2) 122.0 (CH) 131.6 (qC) 18.1 (CH3) 25.7 (CH3) 29.2 (CH2) 137.9 (CH) 127.7 (qC) 171.1 (qC) 20.7 (CH3) 30.4 (CH2) 49.5 (CH) 84.2 (qC) 28.7 (CH3) 30.0 (CH3) 54.1

8 156.4 (qC) 103.0 (qC) 161.1 (qC) 109.0 (qC) 155.7 (qC) 86.5 (qC) 209.7 (qC) 46.6 (CH) 65.6 (CH) 50.0 (CH) 194.1 (qC) 101.8 (qC) 88.3 (qC) 115.3 (CH) 126.3 (CH) 78.4 (qC) 28.2 (CH3) 28.5 (CH3) 21.5 (CH2) 122.6 (CH) 131.0 (qC) 18.1 (CH3) 25.6 (CH3) 27.9 (CH2) 139.7 (CH) 127.0 (qC) 172.9 (qC) 20.5 (CH3) 20.2 (CH2) 42.8 (CH) 82.2 (qC) 27.1 (CH3) 29.7 (CH3)

9 159.1 (qC) 108.0 (qC) 163.1 (qC) 106.1 (qC) 153.8 (qC) 86.3 (qC) 210.0 (qC) 46.3 (CH) 65.4 (CH) 49.7 (CH) 194.0 (qC) 101.8 (qC) 88.2 (qC) 21.0 (CH2) 121.5 (CH) 137.1 (qC) 39.6 (CH2) 26.3 (CH2) 123.9 (CH) 131.4 (qC) 25.5 (CH3) 17.5 (CH3) 16.0 (CH3) 21.8 (CH2) 121.6 (CH) 135.2 (qC) 17.8 (CH3) 25.6 (CH3) 27.6 (CH2) 140.5 (CH) 126.1 (qC) 172.6 (qC) 20.4 (CH3) 20.0 (CH2) 42.6 (CH) 82.0 (qC) 27.0 (CH3) 29.5 (CH3)

10 157.4 (qC) 102.0 (qC) 161.3 (qC) 107.3 (qC) 157.7 (qC) 83.7 (qC) 203.8 (qC) 47.1 (CH) 134.7 (CH) 133.7 (qC) 179.0 (qC) 100.5 (qC) 90.6 (qC) 116.0 (CH) 123.3 (CH) 81.2 (qC) 45.0 (CH2) 121.1 (CH) 141.4 (CH) 71.8 (qC) 30.1 (CH3) 29.1 (CH3) 28.7 (CH3) 21.7 (CH2) 122.3 (CH) 131.2 (qC) 18.2 (CH3) 25.8 (CH3) 29.7 (CH2) 136.2 (CH) 128.7 (qC) 168.8 (qC) 20.9 (CH3) 25.3 (CH2) 48.9 (CH) 83.8 (qC) 29.0 (CH3) 30.0 (CH3)

11 163.1 (qC) 105.1 (qC) 167.4 (qC) 103.8 (qC) 152.9 (qC) 84.1 (qC) 203.0 (qC) 46.8 (CH) 134.4 (CH) 133.7 (qC) 178.1 (qC) 100.7 (qC) 90.0 (qC) 21.2 (CH2) 121.5 (CH) 135.4 (qC) 39.7 (CH2) 26.7 (CH2) 124.2 (CH) 131.3 (qC) 25.7 (CH3) 17.6 (CH3) 16.1 (CH3) 26.8 (CH2) 91.3 (CH) 71.6 (qC) 24.2 (CH3) 25.8 (CH3) 29.6 (CH2) 137.5 (CH) 129.1 (qC) 171.8 (qC) 20.6 (CH3) 25.0 (CH2) 48.7 (CH) 84.0 (qC) 28.9 (CH3) 30.0 (CH3)

12

54.0

157.4 (qC) 103.1 (qC) 161.2 (qC) 108.5 (qC) 157.5 (qC) 83.8 (qC) 201.6 (qC) 84.9 (qC) 134.7 (CH) 132.2 (qC) 179.6 (qC) 100.8 (qC) 89.2 (qC) 115.4 (CH) 126.4 (CH) 78.8 (qC) 28.3 (CH3) 28.3 (CH3) 21.6 (CH2) 121.8 (CH) 132.0 (qC) 18.2 (CH3) 25.8 (CH3) 28.3 (CH2) 118.0 (CH) 138.3 (qC) 12.6 (CH3) 68.0 (CH2) 30.1 (CH2) 49.7 (CH) 84.2 (qC) 29.0 (CH3) 30.2 (CH3)

Cytotoxic Polyprenylated Xanthones from Garcinia hanburyi Journal of Natural Products, 2009, Vol. 72, No. 1 119

120 Journal of Natural Products, 2009, Vol. 72, No. 1

Figure 1. Key COSY (bold lines) and HMBC (HfC) of fragments of 1.

Figure 2. Key ROESY (HTH) correlations of 1. Compound 3 was obtained as a yellow gum. The HREIMS established the molecular formula as C38H48O6 ([M]+ at m/z 600.3464, calcd 600.3451), possessing 15 degrees of unsaturation. The 1H NMR spectrum of 3 (Table 1) revealed the presence of one hydrogen-bonded hydroxy group [δH 12.76 (1H, s, 1-OH)], Scheme 1. Postulated Biosynthetic Pathway Leading to 1

Tao et al. four prenyl olefinic protons [δH 4.40 (1H, dt, J ) 1.2, 7.2 Hz), 5.09 (1H, dt, J ) 1.2, 7.2 Hz), 5.20 (1H, dt, J ) 1.2, 7.2 Hz), 5.44 (1H, dt, J ) 1.2, 7.2 Hz)], an olefinic proton [δH 7.45 (1H, d, J ) 7.2 Hz)], an aromatic proton [δH 6.10 (1H, s)], and nine methyl groups [δH 0.99, 1.28, 1.37, 1.61, 1.66, 1.68, 1.71, 1.72, 1.72 (each 3H, s)]. Comparison of the NMR data of 3 and forbesione (3a) revealed the only difference was that the hydroxy group at C-3 in 3a was replaced by a 3-O-geranyl group in 3,14 which could be confirmed by the HMBC correlations of H-11/C-3, C-12, and C-13 and the ROESY correlations of H-2/H-11. Thus, compound 3 was assigned as 3-O-geranylforbesione. Compound 4 was isolated as a yellow gum. Its molecular formula of C38H44O8 was established from HREIMS ([M]+ at m/z 628.3082, calcd 628.3036), with 17 degrees of unsaturation. Comparison of its NMR spectra (Tables 1 and 2) with those of morellic acid15 showed the presence of a 1-methylcyclohexene ring fused with dimethylpyran ring16 via C-11 (δC 32.8) and C-16 (δC 45.0) and the absence of the C11/C16 double bond in 4. The HMBC correlations of H-12/C-2, C-11, C-14, C-16, and C-20 and H-15/ C-11 and C-16 confirmed the fusion of the two rings. The ROESY correlations of H-11/H-16 suggested the cis relative configuration of these two protons. The structure of 4 was thus elucidated as gambogefic acid. Compound 5 was obtained as yellow needles. The molecular formula was assigned as C39H46O9 by HREIMS ([M]+ at m/z 658.3147, cald 658.3142), 30 mass units more than that of gambogellic acid (5a).3 The 1H and 13C NMR data (Tables 1 and 2) of 5 were compared to those of 5a. It was observed that the H-7 signal at δ 3.46 (dd, J ) 4.6, 6.9 Hz) of 5a was replaced by a methoxyl singlet at δ 3.63 in 5. The attachment of a methoxy group to C-7 was indicated by the HMBC correlations from its protons to C-7. The olefinic proton doublet at δ 7.49 [d, J ) 6.9 Hz, (H8)] was also replaced by a singlet at δ 7.56 since it no longer has a coupling partner on C-7. Thus, the planar structure of 5 was established as 7-methoxygambogellic acid. Compounds 6 and 7 were isolated as a yellow gum and gave the same molecular formula C39H46O9 by their HREIMS ([M]+ at

Cytotoxic Polyprenylated Xanthones from Garcinia hanburyi

Journal of Natural Products, 2009, Vol. 72, No. 1 121

Figure 3. Key ROESY (HTH) correlations of 8.

m/z 658.3133 and 658.3135, cald 658.3142). Both compounds displayed similar NMR features (Tables 1 and 2) and the same fragmentation patterns in the EIMS, suggesting that 6 and 7 were isomers. The UV absorption bands at λmax 365 and 290 nm were similar to those of R-gambogic acid (6a) and S-gambogic acid (7a).17 Spectroscopic analyses revealed that they were 7-methoxylated derivatives of 6a and 7a. The HMBC correlations from the methoxy group to C-7 confirmed the assignment. In the previous report, the single crystal of 6a had been obtained from recrystallization of the pyridine salt of gambogic acid, and its stereochemistry was then determined by X-ray crystallographic analysis.18 According to the literature, the methoxy group substitution at C-7 only changed the chemical shifts and peak patterns of the neighboring atoms, such as desoxymorellin19 and 7-methoxydesoxymorellin,7 forbesione,14 and gaudichaudione H,20 as well as gambogellic acid3 and 5. Therefore, the configurations at C-13 in 6 and 7 were determined as R and S, respectively, by comparing the NMR spectroscopic patterns with those of 6a and 7a.17 On the basis of the above data, the structures of 6a and 7a were assigned as 7-methoxygambogicacidand7-methoxyepigambogicacid,respectively.

Compound 8 was isolated as a yellow gum. Its molecular formula was established as C33H38O9 on the basis of HREIMS at m/z 578.2523 ([M]+, calcd 578.2516), 18 mass units more than that of morellic acid.15 The UV absorption bands at λmax 364, 316, 276, and 266 nm were similar to those of 2a.3 Analysis of the NMR data (Tables 1 and 2) of 8 and 2a showed that these two compounds are similar except for the presence of a hydroxy group at C-8 in 8 instead of a methoxy group in 2a.3 The relative configuration of 8 was assigned the same as that of 2a by comparing their NMR data and the ROESY correlations of H-8/H-7, H-8/H-8a, and H-8/H26b (Figure 3). Thus, the structure of 8 was established as 8,8adihydro-8-hydroxymorellic acid. Compound 9, a yellow gum, was assigned a molecular formula of C38H48O9 from its HREIMS ([M]+ at m/z 648.3323, calcd 648.3298), 18 mass units more than that of gambogenic acid.3 Analysis of its 1D and 2D NMR spectra showed that the only difference between 9 and gambogenic acid was the presence of a hydroxy group at C-8 in 9. ROESY correlations of H-8/H-7, H-8/ H-8a, and H-8/H-31b revealed that 9 has the same relative configuration as that of 2a. Therefore, the structure of 9 was assigned as 8,8a-dihydro-8-hydroxygambogenic acid. Compound 10 had a molecular formula of C38H44O9, established by HREIMS ([M]+ at m/z 644.2965, calcd 644.2985), 16 mass units more than that of gambogic acid.17 Detailed analysis of the NMR spectra of 10 and gambogic acid showed the presence of 3-hydroxy3-methlbut-1-enyl instead of 3-methylbut-2-enyl group substitution at C-14, which was confirmed by the HMBC correlations of H-14/ C-15 and C-16, H-16/C-17, H-18/C-16 and C-17, and H-19/C-16 and C-17. The configuration of the C15/C16 double bond was not clear owing to the overlapped signals of H-15 and H-16. Consequently, compound 10 was defined as oxygambogic acid. Compound 11 exhibited a molecular formula of C38H46O9, determined by HREIMS, which was 16 mass units more than that of gambogenic acid, implying one more oxygen atom in 11. The 1H NMR, 13C NMR, HMBC, and ROESY spectra revealed the presence of one 2-(2-hydroxypropyl)dihydrofuran ring [δH 2.93 (dd, J ) 7.6, 15.2 Hz, Hb-21), 3.10 (dd, J ) 10, 15.2 Hz, Ha-21), 4.55 (t, J ) 8.8 Hz, H-22), 1.14 (s, H3-24), and 1.23 (s, H3-25)] attached to C-3 and C-4.21 The HMBC correlations from H-21 to C-3, C-4, C-4a, and C-22 and from H-24, H-25 to C-22 and C-23 supported the structure assignment (Tables 1 and 2). The relative configuration at C-22 was assigned the same as that of cantleyanone B by the ROESY correlations of H-21a/H-22, H-21a/H-30, H-21b/H-24, H-21b/H-25, and H-21b/H-35.21 Compound 11 was named gambogenific acid. Compound 12 exhibited a molecular formula of C34H40O8 by HREIMS ([M]+ ion at m/z 576.2707, calcd 576.2723), 30 mass units more than that of isomorellinol.2 Spectroscopic analyses revealed that the only difference between 12 and isomorellinol was

Tao et al.

0.63 ( 0.02 0.64 ( 0.02 1.21 ( 0.05 1.79 ( 0.14 1.91 ( 0.17 5.08 ( 0.92 1.71 ( 0.14 0.85 ( 0.10 0.93 ( 0.09 0.91 ( 0.02 0.56 ( 0.03 4.09 ( 0.37 111 ( 4.58 8.68 ( 0.18 IC50 (µM)a

Inhibitory activity was expressed as the mean ( SEM of 50% inhibitory concentration of triplicate determinations and was obtained by interpolation of concentration-inhibiton curves.

7 6 5 4 3 2 1

Table 3. Cytotoxicity of Compounds 1-14 Against HeLa Cells

General Experimental Procedures. Optical rotations were determined on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Shimadzu UV-2450 spectrometer. IR spectra were recorded on a Perkin-Elmer 577 spectrometer. NMR spectra were measured on a Bruker AM-400 spectrometer for 1H, 13C, HSQC, and HMBC and a Varian Inova-600 spectrometer for COSY and ROESY. The pulse conditions were as follows: for 1H, spectrometer frequency (SF) 400.16 MHz, acquisition time (AQ) 2.048 s, relaxation delay (RD) 1.0 s, spectral width (SW) 8000 Hz, and FT size 32 768 data points; for 13C, SF 100.639 MHz, AQ 0.803 s, RD 1.0 s, SW 20 408.1 Hz, line broadening 2.0 Hz, and FT size 32 768; for HSQC, AQ 0.341 s, RD 1.0 s, SW 6008.6 Hz (1H) and 21 978.0 Hz (13C), number of points (NP) 4096, number of increments (NI) 2 × 200, FT size 2048 × 4096; for HMBC, AQ 0.205 s, RD 1.0 s, SW 5000.0 Hz (1H) and 22 988.5 Hz (13C), NP 2048, NI 300, FT size 2048 × 2048; for COSY, AQ 0.128 s, RD 1.0 s, SW 8000.0 Hz, NP 2048, NI 320, FT size 2048 × 2048; for ROESY, AQ 0.213 s, RD 1.0 s, SW 10 999.6 Hz, NP 2048, NI 2 × 512, mixing time 0.230 s. EIMS (70 eV) were carried out on a Finnigan-MAT 95 mass spectrometer, and ESIMS was carried out on a Finnigan LCQDECA instrument. Analytical and semipreparative HPLC was performed on an Agilent 1100 with an Agilent DAD spectrophotometer and a SB-C18 column (4.6 × 250 mm, 5 µm) or Exlipse XDB-C18 column (10 × 250 mm, 5 µm). All solvents used were of analytical grade (Shanghai Chemical Reagents Company, Ltd.). Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Ltd.) and Sephadex LH-20 gel (Amersham Biosciences) were also used for column chromatography. Plant Material. The gamboge resin of G. hanburyi was purchased in Shanghai, China, in September 2007. The voucher specimen (SC0091009) was identified by one of the authors (D.-A.G.) and deposited in Shanghai Research Center for TCM Modernization. Extraction and Isolation. The dried gum resin of G. hanburyi (800 g) was powdered and extracted with acetone (3 × 4 L) at room temperature for two days. The acetone extract (550 g) was chromatographed on a silica gel column eluted successively with a petroleum ether/acetone gradient (100:0 to 0:100) to obtain seven fractions. Fraction 2 (5 g) was subjected to a silica gel column eluting with petroleum ether-EtOAc (30:1 to 4:1) to afford six major fractions, F2a-F2f. F2c was then subjected to Sephadex LH-20 (petroleum ether-CHCl3-MeOH, 2:1:1) to afford three major fractions, which were further purified by semipreparative HPLC (ACN-H2O, 94:6) to afford 1 (5 mg), 2 (3 mg), desoxymorellin (56 mg), desoxygambogenin (200 mg), 3 (7 mg), gambogin (12.4 mg), and its diastereoisomer (11.8 mg). Fraction 3 (2 g) was subjected to column chromatography over

8

9

Experimental Section

a

10

11

12

13

14

R-gambogic acid

an additional methoxy group located at C-7 in 12. Therefore, compound 12 was identified as 7-methoxyisomorellinol. Compounds 13 and 14 gave the same molecular formula, C38H46O9, which was established by their HREIMS, showing 18 mass units more than that of gambogic acid.17 Both compounds displayed similar NMR spectroscopic features and the same fragmentation patterns in the EIMS, suggesting that 13 and 14 were isomers. The UV absorption bands of 13 and 14 were similar to those of 8. Spectroscopic analyses revealed that they were 8-hydroxylated derivatives of gambogic acid. Thus, compounds 13 and 14 were established as 8,8a-dihydro-8-hydroxygambogic acid and its isomer, respectively. However, the configuration of C-13 in 13 and 14 has not been identified yet. The structures of known compounds were identified as desoxymorellin,19 desoxygambogenin,3 gambogin and its diastereoisomer,3 isomorellin,3 isogambogenin,3 hanburin,3 gambogellic acid (5a),3 gambogenic acid,3 morellic acid,15 gambogic acid (6a),17 epigambogic acid (7a),17 isogambogic acid,2 epiisogambogic acid,2 isomorellinol,2 gaudichaudic acid,5 isogambogenic acid,5 and isomorellic acid,15 respectively, by comparison of their spectroscopic data with those of related literatures. All isolated compounds were evaluated for their cytotoxicity against HeLa human cervical carcinoma cells, with adriamycin as the positive control. All but compound 2 displayed potent cytotoxicity. The IC50 values of the new compounds (1-14) and R-gambogic acid are given in Table 3.

0.70 ( 0.08

122 Journal of Natural Products, 2009, Vol. 72, No. 1

Cytotoxic Polyprenylated Xanthones from Garcinia hanburyi Sephadex LH-20 (petroleum ether-CHCl3-MeOH, 2:1:1) and silica gel (petroleum ether-Me2CO, 20:1) to obtain four major fractions, F3a-F3d. F3b was further purified by semipreparative HPLC (ACN-H2O, 94:6) to afford isomorellin (40 mg) and isogambogenin (60 mg). F3c was further purified by recrystallization from CHCl3-Me2CO (5:1) to obtain hanburin (20 mg). Fraction 4 (8 g) was chromatographed on a silica gel column (CHCl3-MeOH, 100:0 to 10: 1) to obtain five major fractions, F4a-F4e. F4e was further purified by semipreparative HPLC (MeOH-H2O-0.08% CF3COOH, 89:9:2) to afford 4 (5 mg), gambogellic acid (35 mg), 5 (4 mg), 6 (18 mg), and 7 (15 mg). Fraction 5 (380 g) was separated on a silica gel column eluting with CHCl3-Me2CO (100:0 to 5:1) to afford six major fractions, F5a-F5f. F5e was separated on Sephadex LH-20 (petroleum ether-CHCl3-MeOH, 2:1:1) to obtain five major fractions, which were further purified by semipreparative HPLC (MeOH-H2O-0.08% CF3COOH, 95:3:2) to obtain gambogenic acid (30 g), morellic acid (4 g), and gambogic acid epimers (80 g). Gambogic acid epimers (100 mg) were subjected to semipreparative HPLC (MeOH-H2O-0.08% CF3COOH, 87:11:2) to afford gambogic acid (40 mg) and epigambogic acid (30 mg). F5f was subjected to column chromatography over silica gel and Sephadex LH-20 (petroleum ether-CHCl3-MeOH, 2:1:1) to obtain five major fractions, which were further purified by semipreparative HPLC (MeOH-H2O-0.08% CF3COOH, 90:8:2) to get 8 (30 mg), 9 (250 mg), 13 (250 mg), 14 (230 mg), and isogambogic acid (500 mg). In the same manner, fraction 6 (70 g) was separated on a silica gel column eluting with CHCl3-MeOH (100:0 to 4:1) to afford four major fractions, which were further purified by semipreparative HPLC (MeOH-H2O-0.08% CF3COOH, 86:12:2) to obtain 10 (8 mg), 11 (16 mg), 12 (9 mg), isomorellinol (20 mg), epiisogambogic acid (100 mg), gaudichaudic acid (10 mg), isogambogenic acid (300 mg), and isomorellic acid (250 mg). Gambospiroene (1): yellow gum; [R]20D +36.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 351 (3.69), 307 (4.27) nm; IR (KBr) νmax 3423, 2966, 2918, 2850, 1730, 1708, 1633, 1616, 1448, 1367, 1174, 1103, 1016, 937 cm-1; 1H and 13C NMR, see Tables 1 and 2; positive ESIMS m/z 603.2 [M + H]+; HRESIMS m/z 625.3138 [M + Na]+ (calcd for C37H46O7Na, 625.3141). Methyl 8,8a-dihydromorellate (2): yellow gum; [R]20D -49.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 366 (3.25), 314 (3.82), 302 (3.77), 275 (4.32), 266 (4.29) nm; IR (KBr) νmax 3423, 2964, 2923, 1737, 1628, 1440, 1261, 1097, 1022, 800 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 576 [M]+ (100), 561 (48), 529 (38), 489 (13), 463 (13), 287 (38), 231 (26), 215 (14); HREIMS m/z 576.2708 [M]+ (calcd for C34H40O8, 576.2723). 3-O-Geranylforbesione (3): yellow gum; [R]20D -422.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 351 (4.29), 212 (4.74) nm; IR (dry film) νmax 2970, 2925, 1740, 1639, 1593, 1425, 1375, 1307, 1170, 1122 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 600 [M]+ (10), 572 (47), 436 (100), 421 (37), 339 (65), 297 (38), 283 (32), 241 (45), 215 (39), 69 (83); HREIMS m/z 600.3464 [M]+ (calcd for C38H48O6, 600.3451). Gambogefic acid (4): yellow gum; [R]20D -265.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 361 (4.25), 214 (4.71) nm; IR (KBr) νmax 3430, 2975, 2927, 1739, 1691, 1631, 1587, 1434, 1309, 1130 cm-1; 1 H and 13C NMR, see Tables 1 and 2; EIMS m/z 628 [M]+ (12), 600 (12), 473 (18), 431 (7), 355 (8), 297 (8), 69 (100); HREIMS m/z 628.3082 [M]+ (calcd for C38H44O8, 628.3036). 7-Methoxygambogellic acid (5): yellow needles (ACN); mp 146-147 °C; [R]20D -307.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 364 (4.15), 215 (4.45) nm; IR (KBr) νmax 3430, 2929, 1745, 1689, 1637, 1591, 1431, 1379, 1325, 1172, 1118 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 658 [M]+ (4), 630 (100), 585 (15), 504 (53), 503 (49), 447 (17), 421 (15), 353 (20), 277 (19); HREIMS m/z 658.3147 [M]+ (calcd for C39H46O9, 658.3142). 7-Methoxygambogic acid (6): yellow gum; [R]20D -535.0 (c 0.085, CHCl3); UV (CH3OH) λmax (log ε) 365 (4.41), 290 (4.45) nm; IR (KBr) νmax 3430, 2970, 2923, 1747, 1689, 1639, 1595, 1458, 1436, 1334, 1174, 1135 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 658 [M]+ (8), 630 (100), 575 (17), 547 (48), 529 (12), 503 (25), 421 (14), 355 (16), 277 (31), 215 (24); HREIMS m/z 658.3133 [M]+ (calcd for C39H46O9, 658.3142). 7-Methoxyepigambogic acid (7): yellow gum; [R]20D -576.0 (c 0.11, CHCl3); UV (CH3OH) λmax (log ε) 365 (4.35), 290 (4.41) nm; IR (KBr) νmax 3430, 2971, 2927, 1747, 1689, 1645, 1595, 1458, 1436, 1334, 1174, 1135 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS

Journal of Natural Products, 2009, Vol. 72, No. 1 123 m/z 658 [M]+ (7), 630 (100), 575 (19), 547 (50), 529 (13), 503 (27), 421 (17), 355 (18), 277 (35), 215 (26); HREIMS m/z 658.3135 [M]+ (calcd for C39H46O9, 658.3142). 8,8a-Dihydro-8-hydroxymorellic acid (8): yellow gum; [R]20D -34.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 364 (3.55), 316 (4.12), 276 (4.61), 266 (4.58) nm; IR (KBr) νmax 3442, 2977, 2927, 1741, 1689, 1645, 1626, 1458, 1380, 1190, 1124 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 578 [M]+ (2), 560 (100), 545 (45), 532 (72), 517 (71), 405 (89), 391 (30), 363 (47), 349 (42), 307 (33), 287 (59), 245 (42), 231 (37), 215 (29); HREIMS m/z 578.2523 [M]+ (calcd for C33H38O9, 578.2516). 8,8a-Dihydro-8-hydroxygambogenic acid (9): yellow gum; [R]20D -67.0 (c 0.11, CHCl3); UV (CH3OH) λmax (log ε) 351 (3.87), 298 (4.46), 203 (4.90) nm; IR (KBr) νmax 3419, 2972, 2925, 1741, 1689, 1631, 1450, 1375, 1323, 1267, 1155, 1083, 1045 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 630 [M - H2O]+ (100), 602 (27), 507 (45), 479 (36), 351 (43), 295 (50), 253 (57); HREIMS m/z 648.3323 [M]+ (calcd for C38H48O9, 648.3298). Oxygambogic acid (10): yellow gum; [R]23D -337.0 (c 0.095, CHCl3); UV (CH3OH) λmax (log ε) 358 (4.09), 292 (4.17), 204 (4.41) nm; IR (dry film) νmax 3432, 2974, 2927, 1736, 1693, 1633, 1592, 1456, 1437, 1335, 1178, 1140, 1049, 756 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 626 [M - H2O]+ (3), 545 (100), 517 (8), 499 (9), 271 (8), 245 (12), 215 (26), 189 (12); HREIMS m/z 644.2965 [M]+ (calcd for C38H44O9, 644.2985). Gambogenific acid (11): yellow gum; [R]23D -402.0 (c 0.095, CHCl3); UV (CH3OH) λmax (log ε) 358 (4.06), 204 (4.46) nm; IR (dry film) νmax 3458, 2976, 2927, 1737, 1685, 1633, 1593, 1456, 1398, 1338, 1149, 1014, 943, 758 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 646 [M]+ (100), 618 (8), 549 (10), 523 (73), 495 (25), 367 (32), 325 (23), 295 (23), 253 (26), 213 (17); HREIMS m/z 646.3138 [M]+ (calcd for C38H46O9, 646.3142). 7-Methoxyisomorellinol (12): yellow gum; [R]23D -416.0 (c 0.090, CHCl3); UV (CH3OH) λmax (log ε) 358 (3.94), 288 (4.05), 227 (4.07) nm; IR (dry film) νmax 3475, 2976, 2927, 1745, 1633, 1593, 1437, 1335, 1136, 754 cm-1; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z 576 [M]+ (8), 548 (100), 517 (16), 479 (20), 435 (34), 379 (17), 287 (24), 263 (24), 245 (23), 231 (18), 215 (14), 135 (15), 69 (42); HREIMS m/z 576.2707 [M]+ (calcd for C34H40O8, 576.2723). 8,8a-Dihydro-8-hydroxygambogic acid (13): yellow gum; [R]24D -77.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 354 (3.59), 315 (4.04), 277 (4.47), 268 (4.42), 204 (4.90) nm; IR (KBr) νmax 3438, 2972, 2926, 1741, 1689, 1628, 1587, 1456, 1375, 1325, 1176, 1045, 754 cm-1; 1H NMR (CDCl3, 400 MHz) δ 11.86 (1H, s), 6.65 (1H, d, J ) 10 Hz), 6.62 (1H, overlapped), 5.45 (1H, d, J ) 10 Hz), 5.09 (1H, t, J ) 7.2 Hz), 4.98 (1H, t, J ) 6.4 Hz), 4.81 (1H, brs), 3.28-3.12 (5H, m), 2.67 (1H, m), 2.44 (1H, d, J ) 8.8 Hz), 2.06 (2H, m), 1.94 (3H, s), 1.91 (1H, m), 1.76 (1H, m), 1.72 (3H, s), 1.65 (3H, s), 1.62 (3H, s), 1.59 (1H, m), 1.56 (3H, s), 1.39 (1H, m), 1.36 (3H, s),1.34 (3H, s), 1.12 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 209.4, 193.7, 172.5, 161.2, 156.2, 155.5, 139.8, 131.6, 130.9, 126.7, 124.8,, 123.5, 122.3, 115.6, 108.5, 102.6, 101.5, 88.1, 86.3, 82.1, 80.8, 65.4, 49.8, 46.4, 42.6, 41.6, 29.5, 27.7, 27.0, 27.0, 25.4, 25.4, 22.6, 21.2, 20.3, 20.0, 17.9, 17.4; EIMS m/z 628 [M - H2O]+ (20), 600 (15), 545 (100), 517 (23), 499 (7), 473 (13), 245 (9), 215 (14), 189 (6); HREIMS m/z 646.3130 [M]+ (calcd for C38H46O9, 646.3142). 8,8a-Dihydro-8-hydroxygambogic acid isomer (14): yellow gum; [R]24D +4.0 (c 0.10, CHCl3); UV (CH3OH) λmax (log ε) 358 (3.53), 316 (3.99), 277 (4.36), 268 (4.31), 204 (4.39) nm; IR (KBr) νmax 3442, 2972, 2927, 1741, 1689, 1628, 1587, 1456, 1375, 1325, 1178, 1045, 754 cm-1;1H NMR (CDCl3, 400 MHz) δ 11.88 (1H, s), 6.65 (1H, d, J ) 10 Hz), 6.60 (1H, t, J ) 7.2 Hz), 5.43 (1H, d, J ) 10 Hz), 5.04 (1H, t, J ) 7.2 Hz), 4.97 (1H, t, J ) 6.8 Hz), 4.81 (1H, brs), 3.28-3.13 (5H, m), 2.67 (1H, m), 2.45 (1H, d, J ) 8.4 Hz), 2.02 (2H, m), 1.94 (3H, s), 1.91 (1H, m), 1.76 (1H, m), 1.72 (3H, s), 1.64 (3H, s), 1.62 (3H, s), 1.59 (1H, m), 1.55 (3H, s), 1.40 (3H, s), 1.38 (1H, m), 1.34 (3H, s), 1.12 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 209.3, 193.7, 172.3, 161.2, 156.2, 155.5, 139.7, 131.7, 130.9, 126.7, 124.5, 123.4, 122.4, 115.5, 108.4, 102.4, 101.5, 88.1, 86.3, 82.1, 80.9, 65.4, 49.8, 46.4, 42.6, 41.8, 29.5, 27.7, 27.5, 26.9, 25.4, 25.4, 22.5, 21.2, 20.3, 20.0, 17.8, 17.4; EIMS m/z 646 [M]+ (2), 628 (16), 600 (15), 545 (100), 517 (24), 499 (8), 473 (12), 245 (12), 215 (21), 189 (10); HREIMS m/z 646.3132 [M]+ (calcd for C38H46O9, 646.3142).

124 Journal of Natural Products, 2009, Vol. 72, No. 1 Cytotoxicity Assay. Cytotoxicity against HeLa cells was evaluated by using the MTT method according to the protocols described22 with adriamycin as a positive control (IC50 ) 0.41 µM against HeLa cells). Acknowledgment. We acknowledge the Department of Instrumental Analysis of our Institute for measuring the UV, IR, NMR, and mass spectra. Supporting Information Available: 1D and 2D NMR spectra of 1-14. These materials are available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Venkataraman, K. Proc. Ind. Natl. Acad. Sci. 1973, 39A, 365–381. (2) Lin, L. J.; Lin, L. Z.; Pezzuto, J. M.; Cordell, G. A. Magn. Reson. Chem. 1993, 31, 340–347. (3) Asano, J.; Chiba, K.; Tada, M.; Yoshii, T. Phytochemistry 1996, 41, 815–820. (4) Sukpondma, Y.; Rukachaisirikul, V.; Phongpaichit, S. Chem. Pharm. Bull. 2005, 53, 850–852. (5) Han, Q. B.; Wang, Y. L.; Yang, L.; Tso, T. F.; Qiao, C. F.; Song, J. Z.; Xu, L. J.; Chen, S. L.; Yang, D. J.; Xu, H. X. Chem. Pharm. Bull. 2006, 54, 265–267. (6) Han, Q. B.; Yang, L.; Wang, Y. L.; Qiao, C. F.; Song, J. Z.; Sun, H. D.; Xu, H. X. Chem. BiodiVersity 2006, 3, 101–105. (7) Reutrakul, V.; Anantachoke, N.; Pohmakotr, M.; Jaipetch, T.; Sophasan, S.; Yoosook, C.; Kasisit, J.; Napaswat, C.; Santisuk, T.; Tuchinda, P. Planta Med. 2007, 73, 33–40. (8) Feng, F.; Liu, W. Y.; Chen, Y. S.; Guo, Q. L.; You, Q. D. J. Asian Nat. Prod. Res. 2007, 9, 735–741.

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